Germ layers

Categories of tissues

Covering Epithelia
Simple squamous
Simple cuboidal
Simple columnar
Stratified squamous
Stratified columnar
Pseudostratified columnar
Glandular Epithelia
Exocrine glands

Endocrine glands
Pancreas
Merocrine
Apocrine
Holocrine
Connective Tissues
Connective tissue
Collagen

MUST TO KNOW IN HISTOPATHOLOGIC TECHNIQUES

1. Ectoderm
2. Mesoderm
3. Endoderm
1. Epithelial = 3 germ layers
2. Nervous = endoderm
3. Muscular = mesoderm
4. Connective = mesoderm
Bowmans, endothelium, loop of Henle, alveoli
Ducts of glands, walls of thyroid follicles
Gallbladder (nonciliated)
Uterine tube (ciliated)
Skin (keratinized)
Vagina, esophagus, cervix (nonkeratinized)
Male urethra
Female reproductive tract (nonciliated)
Trachea (ciliated), Epididymis
w/ ducts
Tubular = stomach, uterus
Acinar/alveolar = pancreas, salivary glands
Tubuloacinar = prostate
Ductless
Exocrine = enzymes
Endocrine = hormones
No loss of cytoplasm
Goblet cells, sweat glands
w/ cytoplasmic loss
Distal portion is pinched off
Mammary glands
Disintegrating cell and its constituents released
Complete breakdown of cell
Sebaceous gland
Support
Framework
Cells are widely separated
Major ingredient of connective tissues
Stains: VgMMAK
Van Gieson
Mallorys aniline blue
Massons trichrome
Alcian blue
Krajians aniline blue

General Connective Tissues

Loose CT
Whartons jelly (acid MPS)
BM (reticular)
Lymph node (reticular)
Embryo (mesenchyme)
Hypodermis
lec.mt 04 |Page | 255

Dense CT

Dermis
Capsules
Tendons
Stroma of cornea

Special Connective Tissues

Cartilage
Hyaline = trachea
Fibrous = intervertebral discs
Elastic = ear, epiglottis
Bone
Cancellous/spongy/trabecular = epiphysis/ends of long bones
Compact/cortical = diaphysis/shaft
Others
Blood
Lymph
Hematopoietic tissues
Acid mucopolysaccharides
Fixative: Lead fixatives
Stain: Alcian blue
Osteogenesis imperfect
Brittle bone disease
Defective production of collagen
Deposits found in Connective Tissues (Eosinophilic)
Fibrin
Early: yellow
Old: blue
Stains:
Mallorys PTAH
Lendrums MSB
Fibrinoid
Necrotizing vasculitis
Staining reactions identical to fibrin
Mixture of exudates & altered cytoplasmic constituents
Hyaline
Degenerated collagen
Hypertension, atheroma, diabetic kidney
Stain: PAS
Amyloid
TB, leprosy, osteomyelitis
Stains: CoMT
Congo Red
Metachromatic stain
Thioflavine
Muscle Tissues
Smooth
Involuntary
Intestines, blood vessels
Skeletal
Striated, voluntary
Skeletal muscles
Cardiac
Striated, involuntary
Heart
Nervous Tissues
CNS
Brain, spinal cord
PNS
Peripheral nerves
Special receptors
Ear, eye, nose
Inflammation
Inflammation
Latin word: Inflammare (to set afire)
5 Cardinal Signs of Inflammation
1. Rubor
Redness
Blood flow Injury
2. Calor
Heat

lec.mt 04 |Page | 256

3. Tumor

Swelling

4. Dolor

Pain

Fluid extravasation
Sensory nerves
Loss of function
Destruction of functional units
Acute inflammation
Vascular & exudative
---(Tissue)---> Microphages
Subchronic inflammation
Intergrade between acute & chronic
Chronic inflammation
Vascular & fibroblastic
---(Tissue)---> Macrophages
Inflammation according to Characterisics of Exudate
Serous inflammation
Serum/secretions from serosal mesothelial cells (3Ps)
Pulmonary TB
Fibrinous inflammation
Diphtheria, rheumatoid pericarditis
Early stage of pneumonia
Catarrhal inflammation
Hypersecretion of mucosa
Hemorrhagic inflammation Blood + exudates
Bacterial infections & other infections
Suppurative/purulent
inflammation
5. Functio laesa

Retrogressive Changes = Organ/Tissue smaller than normal

Developmental defects: AAHA
Aplasia
Incomplete/defective development of a tissue/organ
Ex. amastia (breast aplasia)
Atresia
Failure to form an opening
Hypoplasia
Failure of an organ to reach its matured size
Agenesia
Complete non-appearance of an organ
Atrophy
Physiologic atrophy
Natural
Thymus, brain, sex organs
Pathologic atrophy
Vascular atrophy
Pressure atrophy
Atrophy of disuse
Exhaustion atrophy
Endocrine atrophy
Brown atrophy
Lipofuscin
Progressive Changes = Organ/Tissue larger than normal
Hypertrophy
Increased tissue size due to increased cell size
Physiologic: size of uterus
Pathologic: Systemic hypertension
Hyperplasia
Increased tissue size due to increased cell number
Physiologic: Glandular proliferation of the female breast, size of uterus (preg.)
Pathologic: Skin warts due to HPV
Compensatory hyperplasia Ex. Enlargement of one kidney
Pathologic hyperplasia
Ex. Endometrial hyperplasia
Congenital hypertrophy
Phenytoin-induced
Degenerative Changes = Tissues have abnormalities
Metaplasia
Reversible
One adult cell type Another adult cell type
lec.mt 04 |Page | 257

Dysplasia
Anaplasia/
Dedifferentiation
Neoplasia/tumor
Oncology
Parts of a tumor
Types of tumor

Benign
Malignant
Leukemia
Lymphoma
Squamous cell papilloma
Squamous cell carcinoma
Hepatoma/
hepatocarcinoma
Melanoma/
melanocarcinoma
Ectopic pregnancy
Grading

Grade
I
II
III
IV
Staging
UICC
AJCS
TNM system
T

Reversible
One type of adult cell Changes in structural components
Irreversible
Criterion toward malignancy
Adult cell More primitive cells (release tumor markers)
Continuous abnormal proliferation of cells w/o control (no purpose/function)
Ex. Leukemia
Study of neoplasm
Tumors
1. Parenchyma = active elements (tumor cells)
2. Stroma = CT framework
1. Capacity to produce death:
- Benign (Ex. mole)
- Malignant
2. Histologic characteristics:
- Medullary = cells (parenchyma) > supporting tissues (stroma)
- Scirrhous = supporting tissues (stroma) > cells (parenchyma)
-oma
SaMe CarE
-sarcoma = mesenchymal/CT
-carcinoma = epithelial tissues
Malignant
Benign
Malignant
Malignant
Malignant
Fallopian tube pregnancy
Grading
Aggressiveness/level of malignancy
Differentiated cells = resemble normal cells
Undifferentiated cells = younger cells
Broders Classification (Grading)
Differentiated Cells
Undifferentiated Cells
75-100%
0-25%
50-75%
25-50%
25-50%
50-75%
0-25%
75-100%
Staging
Size, extent of spread to lymph nodes, +/- metastases
TNM classification
Grading + staging
TNM System
Applicable to all forms of neoplasia
1 tumor
#: denotes the size of tumor and its local extent
Tis = carcinoma in situ
Ta = non-invasive
Tx = cannot be evaluated
T0 = free of tumor

Treatment
Surgery

Radiation

lec.mt 04 |Page | 258

Teratomas

Apoptosis
Necrobiosis
Necrosis
Types of Necrosis
Coagulation necrosis

Liquefaction/colliquative
necrosis
Caseous/caseation necrosis
Gangrenous necrosis
Fat necrosis
Fatty degeneration
1 changes
2 changes
Algor mortis (1st)

T1 = lesion <2 cm (T1a = <0.5 cm | T1b = <1 cm | T1c = <2 cm)

T2 = lesion 2-5 cm (invasion in muscle)
T3 = skin and/or chest wall involved by invasion (T3a = deep muscle | T3b =
through organ)
T4 = tumor invasion/fixation (T4a = adjacent organ | T4b = fixation to bladder or
colonic wall, in breast, edema)
Regional lymph node involvement
High # denotes increasing extent of involvement
Nx = not evaluable
N0 = no axillary nodes involved
N1 = 1 mobile regional (axillary) node involved
N2 = multiple, mobile regional nodes involved
N3 = fixed regional lymph node involved
N4 = beyond regional lymph node involvement
Metastasis
M0 = no evidence of metastases
M1 = distant metastases are present
Mx = distant metastases not evaluable
Compound tumors
Greek: Monstrous tumors
May contain hair, teeth, bones
w/ heartbeat
Cellular Death
Programmed cell death (cellular suicide)
Physiologic cell death
Ex. normal sloughing off of skin cells
Pathologic cell death
Most common
Tombstone formation
MyLKS
Myocardium
Lungs
Kidneys
Spleen
Pus formation
Brain & spinal cord
Yellow, cheesy, crumbly material
TB, syphilis, tularemia, lymphogranuloma inguinale
Sulfide gas production
a. Dry gangrene = arterial occlusion
b. Wet gangrene = venous occlusion
Chalky white precipitates
Pancreatic degeneration
Liver
Somatic death
During somatic death
CRC: circulatory, respiratory, CNS failure
After somatic death
ARLP DPA: Algor mortis, Rigor mortis, Livor mortis, Postmortem clotting,
Dessication, Putrefaction, Autolysis
Postmortem cooling
lec.mt 04 |Page | 259

Cooling: 7F/hr
Rigor mortis
Stiffening
1st: neck & head (2-3 hrs)
Persists for 3-4 days
Livor mortis
Lividity/suggillations
Purplish discoloration
After 10-12 hrs, it does not blanch on pressure or shift when the body is moved
Postmortem Lividity vs. Ecchymosis
Postmortem Lividity
Ecchymosis
Disappears on pressure (reappears when pressure is Opposite of postmortem lividity
released)
Oozing of blood (incision)
No oozing of blood (incision)
Postmortem Clot vs. Antemortem Clot
Postmortem Clot
Antemortem Clot
Settling of RBCs from plasma
Not readily detachable from the blood vessels
Chicken fat
No chicken fat
Currant jelly
No currant jelly
Assumes the shape of the vessel
Seldom assumes the shape of blood vessels
Rubbery consistency
Granular & friable
Dessication
Drying & wrinkling of the anterior chamber of the eye
Putrefaction
Invasion of intestinal microorganisms
Autolysis
Self digestion of cells
Lysosome: suicide sac of the cell, releases lysozyme
Organ Weights
Liver
1,100 1,600g
Brain
1,150 1,450g
Right lung
300-400g
Left lung
250-350g
Heart
250-300g
Spleen
60-300g
Thyroid
10-50g
Adrenals
4g or so each
Exfoliative Cytology
Exfoliative cytology
Desquamated cells
Pap smear stain method
Method of choice
Barr body
PAP smear
3 anatomical sites
1. Upper lateral third of the vagina
2. Ectocervix
(Stratified Squamous Epithelium)
--------------------------------T zone: detect cervical cancer-------------------------------(Simple Squamous Epithelium)
3. Endocervix
50% alcohol = All types
Fixation
50% alcohol = pleural & peritoneal
70% alcohol = sputum
95% alcohol = urine, bronchial & gastric
Saccomannos fixative = 50% ETOH + 2% carbowax
Smear preparation
Fix immediately
2-3 slides/patient
a. streaking
b. spreading
lec.mt 04 |Page | 260
(2nd)

Fixing smear
Sputum

BAL
Jelly-like clots
GI specimen
Urine
Pap smear

Adhesive agents
3 primary materials used
for obtaining specimen for
Pap smear
Strawberry cervix

Shift to the left

Shift to the right
Shift to the midzone
Superficial cells

Intermediate cells

c. touch preparation/impression
d. pull-apart
Never touch the bottom of the fixative container
Equal parts of ethanol & ether = BEST (but highly flammable)
95% ethanol = commonly used
Spray fixatives = 1 ft away
Saccomannos fixative
3 specimen
(+) alveolar macrophage = sputum
(-) alveolar macrophage = saliva
P. carinii/P. jiroveci
Prevent by adding 300U heparin/100mL aspirate
If > hr delay of fixation digestion of cells
Fasting: 8 hrs
50mL = cytology
10-15mL = UA
2nd urine = preferred
Dr. George Papanicolau (1940)
Smears: prepared by rotary motion
1. Mailing of specimens
- air drying after 2 hrs fixation
- glycerin technique
2. Egg albumin = not recommended as adhesive agent (intensifies stain by Light
Green)
Pooled human serum/plasma
Celloidin ether alcohol
Leuconostoc culture
1. Speculum
2. Ayers spatula = rotate 3600
3. Cytobrush = Os
T. vaginalis
Cells (Cervicovaginal Smears)
Parabasal | Intermediate | Superficial
----------Estrogen----------

45-50m
Pyknotic nucleus
True acidophilia
Folds/curls on edges
a. Navicular cells = boat-shaped

Parabasal cells

15-30m
Fried eggs w/ sunny side up

Endometrial cells

Groups of 3 or more
1-10 days after menst
Endocervical glandular cells Honeycomb appearance
Similar appearance to parabasal cells
lec.mt 04 |Page | 261

Doderlein bacillus

L. acidophilus
Paps stain: blue to lavender
Diabetic patients
Sish kebab appearance
Pear-shaped, blue-gray to blue-green
Pigs on a scruff appearance
Indicates T. vaginalis infection
Clue cells
Wrinkled prune appearance
w/ perinuclear halo
HPV (LSIL)
Formation of salt crystals

C. albicans
T. vaginalis
Leptothrix
G. vaginalis
Koilocytes
Ferning

Early pregnancy
Quantitative Evaluation: Cytohormonal Maturation Index (CHMI)
MI = P/I/S

CHMI
Pregnancy
Newborn (8 weeks)
Infancy (8 weeks-puberty)
Late menopausal
75 y.o. woman w/ estrogen
therapy

MI = 100/0/0 (no estrogen)

MI = 0/20/80
Quality Assurance

3 copies/report

1. Doctor
2. Patient = original copy
3. File
Reports
Surgical pathology report
Cytopathology report
Autopsy report
Signatories
Request forms = patients doctor
Result forms = pathologist
Turnover of results
Surgical pathology & cytology = 24 hrs
Frozen section = 5-15 mins
Autopsy report = 1 week (Autopsy procedure: 24 hrs)
Storage
Specimen (tissue) = 1 month to 1 year
Tissue blocks (paraffin) = 3 to 10 years
Slides = indefinite
Suggested Guidelines for Record and Specimen Retention (Henry, 21st Ed.)
Records
Requisitions
2 years
QC
2 years
Instrument maintenance
2 years
BB QC
5 years
BB employee signatures
10 years
BB donor/recipient records Indefinitely
Reports
Clinical pathology lab
2 years
reports
Surgical pathology (and
10 years
BM) reports
Cytogenetics reports
20 years
Autopsy forensic reports
Indefinitely
lec.mt 04 |Page | 262

Specimens
Serum/other body fluids
Blood smears (routine)
Microbiology smears
BB donor/recipient
specimens
Pathology/BM slides
Pathology blocks
Cytogenetic slides
Cytogenetics diagnostic
images
Forensic Cases
Body fluids
Tissue for toxicology
Wet tissues
Paraffin blocks
Slides
Reports
Gross photographs/
negatives
Dried blood films
Frozen tissue for DNA
Autopsy

Types of autopsy

Preliminaries for PME

48 hours
7 days
7 days
7 days post-transfusion
10 years
10 years
3 years
20 years
1 year
1 year
3 years
Indefinitely
Indefinitely
Indefinitely
Indefinitely
Indefinitely
indefinitely
Autopsy (Postmortem Examination)
Gold standard for confirmation of a medical disease
Wherever scientific medicine of high quality is practiced, postmortem exams
are performed
Whenever a conscientious physician knows why he lost his patient, a
postmortem exam has been performed
Whenever criminal law is enforced
Whenever a death certificate shows accurately the causes of death & confirmed
medical diagnosis for the assembling of vital statistics, a postmortem has been
performed
Whenever there is medical research on the causes & nature of diseases such as
cancer, heart diseases & stroke, the investigative method is the postmortem
exam
An informed society requires a postmortem exam in human death for the good
of medical science, for the publics health & for the future care of the living
patient
1. Complete autopsy
- Requires consent
- Complete examination of all organs, including the brain
2. Partial autopsy
- Part of the anatomy
3. Selective autopsy
- Restricted to at least a single organ (Ex. MI heart)
1. Written consent from the next kin-abide by the extent or restrictions allowed
- Relative: oriented by the attending physician, not the pathologist
2. Death certificate (Old: Blue form | New: Blue border/frame)
- Signed by:
a. Physician
b. Pathologist (back): will sign when PME has been performed
3. Medical abstract or clinical data
lec.mt 04 |Page | 263

4. Medico-legal clearance
- Suspicious evidence of foul play
- Ex. physical injury
Other Uses of Death
Certificate

PME is permitted w/o

consent in the following
circumstances

If pathologist is not
available
The coroner has authority
in the following cases

Somatic death
Criteria for the
pronouncement of death

Burial & cremation purposes

Transport of body from hospital funeral cemetery
Medical insurance claiming
- If suicide: (-) insurance
- Acts of God (lightning, flood): (-) insurance
- Civil war: (-) insurance
1. When it is ordered by the police or coroner (NBI)
2. When it is necessary to complete the death certificate
3. When the deceased himself has given consent before he died (advanced
directive)
- Stipulate that in the event you will die, you will be giving out a consent for
autopsy
- Donate your organs for medical purposes or for transplantations
4. Deceased military personnel who dies in active services/training duty or
military services
The medico-legal examiner or the coroner has jurisdiction in medico-legal cases
& may authorize the pathologist to proceed w/ an autopsy
1. All natural deaths occurring in the hospital w/in 24 hrs of admission, unless
the case was attended by a private physician w/in 36 hrs of death
2. Newborns in the 1st 24 hrs of life
3. All injury cases, old or recent
4. All deaths due to unknown cases
5. All deaths due to suspicious cases
6. All abortion, whether self-induced or otherwise
7. All violent deaths
8. All accidental deaths
9. All sudden deaths
10. All cases w/o medical attendance w/in 36 hrs prior to the hour of death
11. All deaths due to drowning, hanging or strangulation (asphyxia)
12. All deaths due to shooting, stab wounds, burns, electricity, lightning,
tetanus, etc.
13. Homicides
14. All suicides
15. All poisoning
16. Stillborn = omission
17. Premature death
Death of an organism
Cessation of circulation & respiration (1960s)
1. Advanced resuscitation techniques that are capable of reviving effectively
cases of clinical death
*Clinical death: cessation of heartbeat & respiration but the brain is still alive
but injured
2. Advance life-sustaining equipment capable of maintaining cardiovascular &
respiratory functions despite severe brain injury
3. Redefinition from cessation to irreversible cessation of cardiorespiratory
functions after resuscitation attempts
4. Brain death: cannot be revived anymore [National institute of neurological
diseases & stroke in the US (1977)]
- Clinically dead & dead are the same
lec.mt 04 |Page | 264

Criteria for brain death

American bar association &

national conference of
commission of uniform
state laws legislative
definition of death (1980)
American academy of
neurology

Postmortem changes

Technique of Virchow

Brain death: perpetual state of deep sleed

a. Coma (patient will not respond) & cerebral unresponsiveness
b. Apnea
c. Absent cephalic (brainstem) reflexes
d. Electrocerebral silence
criteria should be present for 30 mins at least 6 hrs after onset of coma & apnea
1. irreversible cessation of circulation & respiratory functions
2. Irreversible cessation of all functions of the entire brain, including the
brainstem is dead
Death:
1. Coma
2. Absence of the following:
- Motor response
- Pupillary response to light & pupils at mid-position
- Corneal reflexes
- Caloric responses
- Gag reflexes
- Coughing in response to tracheal suctioning
- Sucking & rooting reflexes
1. Algor mortis
- 1st demonstrable change after death is cooling of the body
- At room temp: 2-2.5F/hr (1st hr)
- 1.5-2F/hr (next 12 hrs)
- 1F/hr (next 12-18 hrs)
- As a rule, the body cools at 1.5F/hr (50% of cases)
- Not a reliable indicator as to the time of death
2. Rigor mortis
- Rigidity of the body due to hardening of the skeletal muscles caused by a
series of physiochemical events after death
- ( formation of locking-chemical bodies
between actin & myosin
- This interlocking is fixed & produces rigor mortis w/o shortening of the
muscles
- Sets w/in 2 hrs after death (head & neck)
- Complete w/ 12 hrs
- Persists about 3-4 days
3. Livor mortis (postmortem lividity/hypostasis)
- Blood supply gravitates to the skin vessels w/c becomes toneless & dilate after
circulation ceases
- Becomes evident as early as 20 mins after death
- Fully evident w/in 4-8 hrs
- Tardien spots: petechiae
4. Postmortem clotting of blood
5. Discoloration of tissue
- Abdomen: green
- Formation of sulfur gases (bacteria)
6. Putrefaction
7. Dessication (mummification)
Techniques of Autopsy
Organs removed & dissected individually in the body
Most widely used metohd
lec.mt 04 |Page | 265

Technique of Rokitansky

Technique of Ghon
Technique of Letulle

Autopsy: Larynx Rectum

Teasing/Dissociation
Crushing/squash
preparation
Smear preparation
Frozen section
Freezing of unfixed tissue
Freezing of fixed tissue
Formal (formol) calcium
Commonly used methods of
freezing
Staining methods
(frozen sections)

H&E

Freeze-drying
Freeze-substitution
Cold knife procedure

In-situ dissection in part combined w/ en bloc technique

En bloc:
- By cavity
- Interrelated to each other
- Systemic dissection
- Ex. thoracic cavity (lungs, heart, diaphragm), respiratory system
En bloc technique
En masse technique
En masse:
- All organs of thoracic, abdominal, & pelvic are removed at the same time
- Sweeping of all organs
Very popular, easy to do, convenient
Part of consent: organs should be retained completely or partially
Organs set aside later
Body undertaker of the body
Fresh Tissue Examination
Tissue specimen Watchglass (isotonic solution) BF/PC microscope
Tissue (<1mm) Sandwich bet. 2 slides/coverslip Vital stain
Spread lightly over a slide (wireloop/applicator)
Frozen Section
(-) Fixative
Best frozen section
To localize hydrolytic enzymes & other antigens
Derivative of formaldehyde
Fix at 4C for 18 hrs
Liquid nitrogen = most rapid
Isopentane cooled by liquid nitrogen
CO2 gas
Aerosol sprays
PATH
Polychrome methylene blue
Alcoholic pinacyanol
Thionine
H & E = progressive, no decolorizer
a. Progressive
- w/o decolorizer
- for frozen sections
b. Regressive
- w/ decolorizer (acid-alcohol)
- for routine histology staining
w/o use of any chemical fixative
Quenching: rapid freezing (-160C)
Sublimation: removal of H2O in the form of ice (-40C) vacuum
Similar to freeze drying but:
Frozen tissue Rossmans formula/1% acetone
Dehydrated in absolute alcohol
Any microtome
Uses CO2
Knife: -40 to -60C
Tissue: 5 to -10C
Environment: 0 to -10C
lec.mt 04 |Page | 266

Cryostat procedure
(Cold microtome)
O.C.T. (Optimal Cutting
Temperature)

Steps

Fixation

pH
Temperature
Microanatomical fixatives

Cytological Fixatives

Nuclear fixatives

Cytoplasmic fixatives

Temperature: -18 to -20C

Cryostat: refrigerated cabinet w/ rotary microtome
Best mounting media for cryostat sections

Tissue Processing
FDCIETS SMoL
1. Fixation
(Decalcification)
2. Dehydration
3. Clearing/Dealcoholization
4. Impregnation/Infiltration
5. Embedding/Casting/Blocking
6. Trimming
7. Sectioning/Microtomy
8. Staining
9. Mounting
10. Labeling (slides)
Fixation
st
1 and most critical step
1 aim: preserve cell (life-like)
2 aim: harden & protect tissues
Most important: stabilization of proteins
6.0-8.0
Room temp = Surgical specimen
0 to 4C = EM and Histochem.
General microscopic study of tissues
a. 10% Formol saline
b. 10% NBF
c. Heidenhains SuSa
d. Formol sublimate (formol corrosive)
e. Zenkers solution
f. Zenker-formol (Hellys)
g. Bouins solution
h. Brasils solution
Specific parts of the cell
a. Nuclear fixatives: w/ glacial acetic acid destroys mitochondria & golgi
bodies (pH 4.6)
b. Cytoplasmic fixatives: w/o glacial acetic acid
c. Histochemical fixatives: preserves chemical constituents
BFNCH
Bouins
Flemmings w/ acetic acid
Newcomers
Carnoys
Heidenhains SuSa
HORFF
Hellys
Orths
Regauds
lec.mt 04 |Page | 267

Histochemical fixatives

Aldehyde Fixatives
Formaldehyde

10% Formol saline

10% NBF

Formol-Corrosive
(formol sublimate)
Glutaraldehyde
Karnovskys
paraformaldehydeglutaraldehyde
Acrolein
Formol-calcium
Mercuric Chloride

Heidenhains SuSa
HgCl2
G.HAc
De-zenkerization
Chromate fixatives

Flemmings w/o acetic acid

Formalin w/ post chroming
FANA
10% Formol saline
Absolute alcohol
Newcomers fluid
Acetone
Concentrated solutions should not be neutralized (explosion)
Stock solution: 37-40%
Working solution: 10% (no buffer: unstable)
Formalin pigments:
a. Paraformaldehyde
- White crystalline precipitates
- Due to prolonged standing
- Removed by: 10% METOH/filtration
b. Acid formaldehyde hematin
- Brown/black granular deposits that may obscure microscopic details
CNS
Best general tissue fixative
Best fixative for tissue containing iron granules
w/ double phosphate buffer
1 mm/hr = rate of tissue penetration
w/ HgCl2
EM
EM: electron histochemistry & electron immunocytochemistry
Mixture w/ formaldehyde/formaldehyde
Lipids (frozen section)
Fixatives
Tissue photography
For Trichrome stain (excellent)
Produce black granular deposits except SuSa
BOSCHZZ
a. B5 = for BM biopsies
b. Ohlmachers
c. Schaudinns
d. Carnoy-Lebrun
e. Heidenhains SuSa = (-) black pigments
f. Zenkers = recommended for trichrome staining
g. Zenker-formol (Hellys) = pituitary gland, BM, & blood containing organs
Su = sublimat (HgCl2)
Sa = saure (acid)
Shrinks tissues
Swells tissues, counteracts HgCl2
Removal of mercuric deposits
H2O I2 H2O Sodium thiosulfate H2O
ROCK
a. Regauds (Mollers) = chromatin, mitochondria, mitotic figures
b. Orths = for Rickettsia, tissue necrosis
lec.mt 04 |Page | 268

Chromate pigments
Lead fixatives
Picric acid fixatives

Glacial acetic acid

Alcoholic fixatives

Osmium tetroxide
(Osmic acid)

Trichloroacetic acid
Acetone
Heat fixation

2 fixation
Post-chromatization
Washing out

EM fixatives

Stains (EM)

c. Chromic acid = preserves CHO

d. K2CrO4 = mitochondria (if acidified, fixes chromatin bodies & chromosomes
but destroys mitochondria)
Fine, yellow brown
Used in 4% aqueous solution of basic lead acetate
For acid MPS and mucin
Highly explosive when dry
Excessive yellow staining of tissues
Picrates Protein Ppt. (H2O soluble) Add 70% ETOH Insoluble
Never wash in H2O before dehydration
For glycogen (excellent)
a. Bouins = for embryos, Massons trichrome stain, glycogen
b. Brasils alcoholic picroformol = less messy than Bouins, glycogen (excellent)
Solidifies at 17C
Fixes & precipitates nucleoproteins, chromosomes, & chromatin material
Most commonly combined w/ other fixatives
Disadvantage: polarization (glycogen granules poles/ends of the cells)
MEICAN
a. Methanol = BM & blood smears
b. Ethanol = preserves but does not fix glycogen (Disadv: polarization)
c. Isopropanol = for touch preparations
d. Carnoys = most rapid (1-3 hrs) | for chromosomes | Dx: rabies (acetone)
e. Alcoholic formalin (Gendres) = sputum
f. Newcomers = for MPS | nuclear & histochemical fixative
Inhibits hematoxylin
Produce black precipitate crystals (osmium oxide)
For lipids
a. Flemmings = permanently fixes fat, for nuclear structures (excellent)
- Fixative & decalcifying agent (chromic acid)
b. Flemmings w/o acetic acid = for mitochondria
Precipitates proteins
Swelling effect counteract shrinkage by other fixatives
Weak decalcifying agent (softening effect)
Recommended for H2O-diffusible enzymes (phosphatases, lipases)
Rabies
Bacteriologic smears
Microwave: 45-55C
Underheating: poor sectioning
Overheating (>65C): vacuolation, overstained cytoplasm
Placing an already fixed tissue in a 2nd fixative
Primarily fixed tissue 2.5-3% K2CrO4 (mordant)
Removing excess fixative
a. Tap H2O = remove excess chromates, formalin, osmic acid (NOT Bouins)
b. 50-70% alcohol = wash out excess picric acid (Bouins)
c. Alcoholic I2 = remove excess mercuric fixatives
Glutaraldehyde
PtCl3
PtCl3 formalin (Zambonis)
AuCl
Osmium tetroxide
10% NBF = acceptable but not recommended
PUL
lec.mt 04 |Page | 269

1. PTA = 1st general stain

2. Uranyl acetate = Best
3. Lead
Factors that Affect Fixation of Tissues
Retarded by:
Size & thickness
(+) Mucus

Prevents complete penetration of fixative

Wash w/ NSS
Fatty tissues: cut in thin sections, fixed longer
Flush out w/ NSS fix
Inactivates enzymes

(+) Fat
(+) Blood
Cold temperature
Enhanced by:
Size & thickness
Agitation
Automatic/mechanical tissue processing
Moderate heat
37-56C
Principles and Precautions in Handling and Fixation of Specimens in General
Autopsy materials
Fixed ASAP
If not possible mortuary refrigerator (4C) or arterial embalming
Surgical specimens
Fixed ASAP
If not possible refrigerate
If placed in NSS during
Autolysis may occur before fixation
operation
If tissues are refrigerated
Avoid slow freezing (ice crystal formation)
Repeated freezing & thawing destroy organelles, release enzymes
Not more than 5mm thick
Size of tissues
Except lung edema: 1-2 cm thick
20:1
Ratio of fixative to tissue
Except osmium tetroxide (expensive) = ratio is 5-10:1
50-100:1
Ratio of fixative to tissue in prolonged fixation (ex. museum preparation)
Avoid drying of small tissue To prevent: place in a petri dish w/ moistened filter paper
biopsies
Hollow organs
Stomach, intestines
Packed w/ cotton soaked fixative or completely opened before being immersed
in adequate fixing solution
Air-filled lungs
Float on fixative
To prevent: cover w/ several layers of gauze to maintain it under surface
Human brains
Suspended by a cord tied under the Circle of Willis to prevent flattening
Avoid Ringers lactate for washing out of blood intravascular perfusion
Fixation time: 2 weeks
Eyes
Not dissected before fixation tissue collapse & wrinkling (escape of vitreous
humor)
Inject formol-alcohol before immersing the organ in the fixative
Glycogen-containing tissues Do not use water
Glycogen is water-soluble
Hard tissues
Cervix, uterus, fibroids, hyperkeratotic skin, fingernails
Wash in running water overnight immerse in 4% aqueous phenol for 1-3
days (Lendrums method)
Difficulties Encountered because of Improper Fixation
Problem
Cause
Failure to arrest early cell autolysis
Failure to fix immediately (tissue was allowed to dry
before fixing)
Insufficient fixative
lec.mt 04 |Page | 270

Removal of substances soluble in fixing agent

Wrong choice of fixative
Presence of artifact pigments on tissue sections
Incomplete washing of fixative
Tissues are soft & feather-like in consistency
Incomplete fixation
Loss/inactivation of enzymes needed for study
Wrong choice of fixative
Shrinkage & swelling of cells & tissue structure
Overfixation
Tissue blocks are brittle & hard
Prolonged fixation
An incompletely fixed tissue may lead to improper & incomplete clearing & impregnation, and may later
prove to be a hindrance to normal sectioning & staining of specimen
Pigment
Acid formaldehyde hematin

Mercuric chloride pigment

Chromate pigment
Osmium tetroxide pigment
Crush artifact
20:1
37C
55C
RT (18-30C)
24-48 hrs
Decalcifying agents

HNO3

5% Formic acid
HCl (Von Ebners)
EDTA
Ion exchange resins
Electrophoresis
Measuring extent of
decalcification
Post-Decalcification
Tissue softeners

Color
Brown/black granules

Removed by:
SAKaL
a. Saturated picric acid
b. Alcoholic KOH
c. Kardasewitsch method
d. Lillies method
Black granules
Alcoholic iodine
Fine, yellow brown
Acid-alcohol
Black precipitate crystals
Cold H2O
Intense eosinophilic staining at the center of the tissue (H & E)
Due to partial coagulation of partially fixed protein
Decalcification
Ratio of decalcifying agent to tissue
Impaired nuclear stain by Van Giesons stain
Tissue Digestion (24-48 hrs)
Optimum temperature
Time
Acids
Chelating agents (EDTA/versene)
Ion exchange resins
Elec. ionization (electrophoresis)
Most common
a. Perenyis = tissue softener & decalcifying agent
b. Phloroglucin-HNO3 = most rapid
- Disadvantage: Yellow color on tissue (neutralize w/ sodium thiosulfate)
Both fixative & decalcifying agent
Best general decalcifying agent
For small pcs of bones & teeth
For small pcs of bones & teeth
For surface decalcification (HCl)
For EM, IHC, & enzyme staining
Hastens decalcification by removing calcium ions from formic acid-containing
decalcifying solutions
Ca2+ are attracted to negative electrode (cathode)
Physical method
Chemical method = CaOx test (routine) | Turbidity = (+) Ca2+
X-ray = most ideal, most sensitive, most reliable but very expensive
- X-ray paper = Kodak X-omat or Faxitron
Removal/neutralization of acid from the tissues after decalcification
Lithium carbonate or sodium bicarbonate solution
4% phenol
Molliflex = tissues appear swollen & soapy
2% HCl
lec.mt 04 |Page | 271

Dehydration
10:1
Ethanol
Methanol
Butanol
Denatured alcohol
Acetone
Dioxane
(Diethylene dioxide)
Tetrahydrofuran (THF)
Graupners method
Weisebergers method
Cellosolve
Triethyl phosphate
Additives to dehydrating
agents
Methods of determining
incomplete dehydration
Xylene (Xylol)
Toluene

Chloroform
Benzene
Methyl salicylate
Methyl benzoate
Cedarwood oil
Clove oil
CCl4
Aniline oil
Glycerin
Gum syrup
Others

1% HCl in 70% alcohol

Dehydration
Aim: To remove fixative & H2O
Ascending grades of alcohol (Start: 65%)
Embryonic & animal tissues: 30% ETOH
Ratio of dehydrating agent to tissue
Best dehydrating agent
Blood & tissue films
Plants & animals
Ethanol + methanol
Both fixative & dehydrating agent
Both dehydrating & clearing agents
Dehydration w/ dioxane
Ethylene glycol monoethyl ether
Combustible and toxic
-a. 4% phenol + 95% ETOH = softener
b. Anhydrous CuSO4 (Last ETOH bath)
- both dehydrating agent & indicator of H2O content of 100% ETOH
- (+) H2O = White Blue
1. Anhydrous CuSO4 method
2. Xylene Milky
Clearing
Most commonly used
Clearing time: to 1 hr
Block size: <5mm
Substitute for xylene/benzene
Clearing time: 1-2 hrs
Not carcinogenic
Toxic fumes
Toxic to liver
For clearing tough tissues
Does not make tissue translucent but removes alcohol
For urgent biopsies
Minimum shrinkage
Aplastic anemia
For double embedding techniques
For CNS, smooth muscles, skin
Minimum shrinkage
Has tendency to become adulterated
Similar to chloroform but is cheaper
Disadvantage: similar to chloroform
For delicate tissues, embryos and insects
No dealcoholization but make the tissues clearer
Citrus fruits oil
Trichloroethane & petrol
Impregnation
lec.mt 04 |Page | 272

25:1
Medium

Paraffin

Paraffin filters
Paraffin oven

Ratio of infiltrating medium to tissue

Paraffin wax
Celloidin (collodion)
Gelatin = H2O soluble, not a wax
Plastic = EM
Introduced by Btschlii
Not recommended for fatty tissues
Low MP = paraffin is soft
High MP = paraffin is hard
Manual: At least 4 changes of wax at 15mins interval
Filtration at 2C above the MP of wax
Ex. Greens no. 904 (coarse filter paper)
2-5C above the melting point of wax (55-60C)
>60C = shrinkage & hardening of tissues

Substitutes for Paraffin Wax

Paraplast
56-57C MP
More elastic & resilient
Bones & brain
Embeddol
56-58C MP
Bioloid
Eyes
Tissue mat
Contains rubber
Ester wax
46-48C MP
Soluble in 95% ETOH & clearing agent
Impregnation w/o prior clearing
H2O soluble wax
38-42C/45-56C MP
Mostly PEG
Carbowax: most commonly used
- Does not require dehydration and clearing
- For enzyme histochemistry
Celloidin/Collodion
Purified form of nitrocellulose
Wet celloidin
Equal parts of ether & alcohol
Bones, brain, teeth sections
Dry celloidin
Gilsons mixture: equal parts of chloroform & cedarwood oil
Whole eye sections
LVN
Another form of celloidin
Soluble in equal concentrations of ether & alcohol
Plastic/Resins
Epoxy (EPON)
Polyester
Acrylic
Waterbath
6-10C below the MP of wax (45-50C)
Autotechnicon
Fixes, dehydrates, clears & infiltrates tissues
Constant tissue agitation
Elliott Bench-Type processor
Wax bath: 3C above the MP of wax
Vacuum embedding
Wax impregnation under negative pressure (hasten removal of air bubbles)
Time is reduced from 25-75% of the normal time required for tissue processing
2-4C above the MP of wax
(+) Odor in the clearing
Indicates that the paraffin wax should be changed
agent
Embedding
Orientation
Arranging in precise positions in the mold, microtome & slide
Oven
5-10C above the MP of wax (Impregnation: 2-5C above)
lec.mt 04 |Page | 273

Blocking-out Molds
Leuckharts embedding
mold
Compound embedding unit
Plastic embedding rings &
basse mold
Tissue Tek
Disposable embedding
molds
Celloidin/Nitrocellulose
method
Double embedding method

Cold knife procedure

Cryostat (Cold microtome)
Fixation
Freeze-drying
Algor mortis
Formol calcium fixation
Impregnation
Embedding
Flotation water bath
Trimming
Routine histopath. (Rotary)
Freezing
EM (Ultrathin)
Rocking/Cambridge
microtome
Rotary/Minot microtome
Sliding microtome

2 L-shaped strips
Adjustable
Several compartments
Special stainless steel base mold fitted w/ a plastic embedding ring (block
holder)
Warm plate
Cold plate (-5C)
1. Peel-away: perfect even block w/o trimming
2. Plastic ice trays: ordinary refrigerators
3. Paper boats: cheap & easy to make
Bones, teeth, eyes
Bell jars: control evaporation
1st: celloidin
2nd: paraffin
Brain
Recall Temperatures
Knife = -40 to -60C
Tissue = 5 to -10C
Environment = 0 to -10C
-18 to -20C
Surgical specimen: room temp
HC & EM: 0-4C
Quenching: -160C
Sublimation: -40C
7F/hr (3.89C/hr)
4C
Manual = 2-5C above MP of wax (55-60C)
Automated = 3C above MP of wax
Vacuum = 2-4C above MP of wax
5-10C above MP of wax
6-10C below MP of wax (45-50C)
Trimming
Removing excess wax after embedding
Ideal: four-sided prism/truncated pyramid
Microtomy
4-6m
10-15m
0.5m
Trefall
Simplest
Minot
Most commonly used for paraffin embedded tissues
Adams
Most dangerous (movable knife)
a. Base-sledge
- For all forms of media
- Block: moving
- Knife: stationary
b. Standard sliding
- Block: stationary
- Knife: moving
lec.mt 04 |Page | 274

Vibrotome
Ultrathin microtome
Freezing microtome
Clearance angle
Bevel angle
Honing
Types of hones
Stropping

Natural dyes

Synthetic dyes
Chromophores
Auxochrome
Chromogen
Dye
Chromophores

Auxochromes
Dye modifiers

Van der Waals forces

Sudanophilia
Hematoxylin

For unfixed, unfrozen tissues

For enzyme demonstration
EM
Diamond knives or broken plate glass
Queckett
Knife to tissue block: 0-150 angle
27-320 angle
Removal of gross nicks
Heel to toe (Edge 1st)
a. Belgium yellow: Best
b. Arkansas
c. Fine carborundum: for badly nicked knives
Removal of gross burrs
Toe to heel (Edge last)
Paddle strop (horseleather)
- Mineral oil = not recommended
- Vegetable oil (castor oil) = applied into the back of the strop, not the surface
Staining
From plants & animals
a. Hematoxylin
b. Cochineal dyes: female Coccus cacti
c. Orcein: from lichens
d. Saffron
A.k.a. coal tar dyes
Derived from benzene & collectively known as aniline dyes
Greek: color-bearer
Coloring property
Greek: increasers
Dyeing property
Benzene + Chromophore
Imparts color temporarily
Chromogen + auxochrome
Imparts color to tissue almost permanently
a. Quinoid ring: Basic fuchsin
b. Azo groups: Congo red
c. Xanthene: Eosin
d. Quinone-imine group
- Oxazin: cresyl fast violet
- Thiazins: toluidine blue
Cationic auxochromes: amino group (NH3+)
Anionic auxochromes: hydroxyl (OH-) and carboxyl (COO-) groups
Attached on benzene ring
a. Ethyl group
b. Methyl group
c. Sulphonic acid
Alum hematoxylin
Tissue stained w/ fat or oil-soluble dyes
H & E Staining
Hematoxylin capechianum/ Hematoxylon campechianum
Nuclear/basic/1 stain
Waldeyer: 1st to use hematoxylin
lec.mt 04 |Page | 275

Lake
Oxidizing agents

Alum hematoxylin

Iron hematoxylin

Tungsten hematoxylin
Copper hematoxylin
Eosin (Eosin Y)

Coplin jar
Slotted staining dishes
Metal/glass staining racks/
carriers
H & E staining steps

Hematoxylin ---(Ripening)---> Hematein (active coloring substance)

Tissue-Mordant-Dye complex
H 2O 2
HgO2 = Harris
K2MnO4
Na perborate
Na iodate = Mayers, Ehrlichs, Gills
Routine H & E = Red
Mordant: K Alum
MEGDH
Mayers = Na iodate (ripening agent)
Ehrlichs = Na iodate (ripening agent)
Gills
Delafields
Harris = HgO2 (ripening agent)
Mordant = oxidizing/ripening agent = Iron
a. Weigerts
- Mordant: FeCl3
- Weigerts + Van Giesons = CT & E. histolytica
b. Heidenhains
- Mordant: Ferric ammonium sulfate
a. Mallorys PTAH
- Mordant = sunlight/K+
- Stain fibrin
Spermatogenesis
Cytoplasmic/acidic/2 stain
Counterstain
a. Eosin Y (Yellowish) = most commonly used
b. Eosin B (Bluish) = deep red
c. Ethyl eosin/Eosin S/Eosin alcohol soluble
Holds 5-9 slides
Holds 5-19 slides
Holds 10-30 slides
1. Xylol (2) = deparaffinization
2. Descending grade of alcohol = rehydration
3. H2O
4. Remove fixative artifact pigments after rehydration & before staining
5. Stain: Nucleus = light blue
6. H2O
7. Acid alcohol (differentiator): Nucleus = light blue
8. Ammonia water (blueing agent): Nucleus = blue
- NH4OH
- LiCO3
- Scotts tap H2O
9. Wash
10. Stain: Eosin Y
11. Ascending grade of alcohol = dehydration
12. Xylene = dealcoholization/clearing
13. Mount & label
Nuclei: blue to blue black
Cytoplasm: pale pink
lec.mt 04 |Page | 276

Pap Smear staining

Benzidine
Acridine orange
Gentian violet
Congo red
Iodine
Malachite green
Janus green
Night blue
Victoria blue
Lysochromes

Adhesives

Mounting media

Stains on skin
Restaining of old sections

Broken slides

Hematoxylin = nuclear stain

OG-6 = cytoplasmic stain (mature superficial cells)
EA (Eosin Azure) = cytoplasmic stain (immature cells: parabasal/intermediate)
EA 65 = for body fluids
EA 36/50 = for gynecologic smears
Other Stains
Hgb
RNA (red fluorescence)
DNA (green fluorescence)
Crystal violet + methyl violet + dextrin
Elastic tissue, amyloid, myelin
Oldest stain
Ascaris eggs
Mitochondria (intravital stain)
Substitute for carbolfuchsin
Neuroglia
Oil soluble dyes
a. SBB = Black (most sensitive)
b. Sudan III = orange
c. Sudan IV (Scharlach R) = red
a. Mayers egg albumin = add thymol crystals (inhibit mold growth)
b. Dried albumin
c. Gelatin
d. Gelatin-formaldehyde
e. Starch paste
f. Plasma
g. Poly-L-lysine = IHC
h. 3-APES: 3-aminopropyltriethoxysilane = Best (cytology)
1.518 = refractive index of glass
1. Resinous media = contains xylene
- a. DPX = 1.532
- b. XAM = 1.52
- c. Canada balsam (Abus balsamea) = 1.524
- d. Clarite = 1.544
2. Aqueous media = for lipids (no xylene)
- Water = temporary mounting, low refractive index
- Glycerin jelly = 1.47 | standard mounting medium (fat stains)
- Gum Arabic (Farrants) = 1.43
- Apathys medium = 1.52
- Bruns fluid = for frozen sections
Others:
- Permount
- HSR
- Clearmount
Remove by using 0.5% acid alcohol tap water
Slide Xylene (24hrs) or gently heat until mounting medium begins to bubble
Remove coverslip Section: Xylene (30mins) H2O 0.5% K2MnO4
(5mins) H2O 5% Oxalic acid (5 mins or til decolorized) H2O Restain
Shortcut: X-XhKhOhR
SlideXyleneRemove coverslipXyleneK2MnO4Oxalic acid Restain
1. Mount the broken slide to another clean xylene-moist slide w/ drop of
lec.mt 04 |Page | 277

mounting media
2. If replacement not possible, the section (if intact) may be transferred to
another slide:
Broken slide Xylene (rem. coverslip) incubate (rem. mountant) 6 parts
butyl acetate + 1 part durofix incubate (mixture film) Cut the film
around the section Cold H2O until the film & section float off Film w/
section mount on a clean slide incubate butyl acetate xylene
mount

Ringing

Enzyme histochemistry
IgG
Polyclonal
Monoclonal
Epithelial Tumor Markers
(+) CK 7
(-) CK 20

Shortcut: Xi6B1DiCuCoFSMiBXM
Broken slide Xylene Incubate 6 Butyl acetate + 1 Durofix Incubate
Cut film Cold H2O to float film & section Film w/ section mount
incubate butyl acetate xylene mount
Sealing the margins of the coverslip
Prevent escape/evaporation of fluid
Immobilize the coverslip
Prevent sticking of slides
a. Kronig cement = 2 parts paraffin + 4-9 parts colophonium resin
b. Durofix (cellulose adhesives)
Immunohistochemistry
Trypsin & protease = most commonly used
Most commonly used antibody
Rabbits (1) > Goat (2) > Pig (3) > Sheep (4) > Horse (5) > Guinea pig (6)
Mice

LUBO = paired
Lung
Uterus
Breast
Ovary
(+) CK 20
Stomach
(-) CK 7
Colon
(+) CK 7
Transitional cell carcinoma of the bladder
(+) CK 20
Mucinous ovarian tumor
(-) CK 7
HCC
(-) CK 20
RCC
SCC
Thyroid carcinoma
Prostatic adenocarcinoma
EMA (Epithelial membrane (+) carcinoma BuLK = paired
antigen)
Breast
Lungs
Kidney
CEA
Oncofetal antigen
GI carcinoma
Differentiates adenocarcinoma (+) & mesothelioma (-)
TTF-1 (Thyroid
Differentiates lung adenocarcinoma & mesothelioma
Transcription Factor)
(+): Thyroid, lung, neuroendocrine tumors
PSA
Prostate cancer
Intermediate Filament Markers
Actin
Smooth muscle
Skeletal muscle
lec.mt 04 |Page | 278

Vimentin
Desmin
GFAP (Glial Fibrillary Acidic
Protein)
NF (Neurofilament)

S100 protein
Neuroendocrine Markers
NSE (Neuron-specific
enolase)
Others

Cardiac muscle
Melanomas
Schwannomas
Leiomyoma (smooth muscle)
Rhabdomyosarcoma (skeletal muscle)
Astrocytoma
Neuroblastoma
Ganglioneuromas
Neuroma
Chemodectoma
Pheochromocytoma
Low MW Ca2+-binding protein
CNS glial cells, Schwann cells
Strong evidence of neural/neuroendocrine differentiation
Chromogranin
Synaptophysin

Germ Cell tumor markers

HCG

Synthesized by syncytiotrophoblasts
Choriocarcinoma
AFP
Endodermal sinus tumors showing yolk sac differentiation
PLAP (Placenta-like ALP)
Germinomas
Mesenchymal Tumor Markers
Myogenic tumors
Myo-D1
Myoglobin
Myogenin
Fibrohistiocytic tumors
-Vascular tumors
Factor VII-related antigen
CD31
UEA: Ulex europaeus I
Melanomas
-Lymphomas
LCA: Leukocyte common antigen (CD45)
Cell Proliferation Markers
Ki67
MIB-1: reference monoclonal antibody for Ki67 demonstration
PCNA
Proliferating cell nuclear antigen
Controls
Positive control
Known
Contains antigen in question
Negative control
Done using a parallel section from the tissue
Internal tissue control
A.k.a. built-in control
Contains the target antigen
Other Topics
Faults During Tissue Processing
Brittle/hard tissue

Clearing agent Milky

Incomplete dehydration
lec.mt 04 |Page | 279

On trimming, tissue smells

of clearing agent
Tissue is opaque
Tissue shrinks away from
wax
Tissue is soft when block is
trimmed
Air holes on tissue
Wax appears crystalline
Paraffin block is moist &
crumbles
Sections fail to form
ribbons

Sections roll up on cutting

adhere & get broken
against the knife edge
Ribbon is curved, crooked,
or uneven
Sections are compressed,
wrinkled or jammed

Sections are squashed

Hole in section
Sections of unequal
thickness
Sections adhere to knife or
other parts of the
microtome

Insufficient impregnation
Insufficient clearing
Insufficient dehydration
Incomplete clearing & impregnation
Incomplete fixation
Incomplete impregnation
Contaminated wax
Block not cooled rapidly enough
Insufficient paraffin impregnation
Surface & edges of block not parallel
Wax too hard
Knife tilted too much
Thick sections
Dull knife
Blunt knife
Dirty knife edge
Irregular knife edge
Edges of block are not parallel
Knife not parallel to the block
Impure paraffin
Blunt/dull knife
Block is warm & soft
Knife edge coated w/ paraffin
Thin sections
Microtome screw is loose
Tilt: vertical
Bevel of knife is lost
Incorrect sharpening
Bubble/dirt
Hard spot in the tissue (Ca2+)
Screw/holder is loose
Large & hard blocks
Static electricity
Dirty knife edge
Dull knife edge

Ribbon is split

Nicks/damage on knife
Dirty embedding
Dirty knife

Chatters are seen

Knife vibrates (hard tissue)

Section: sometimes thin &

thick

Blunt knife

Frozen tissue crumbles &

comes off when the block

Knife/block holder is loose

Inadequate freezing
lec.mt 04 |Page | 280

holder when cut

Frozen tissue chips into
fragments

Tissue is frozen too hard

lec.mt 04 |Page | 281

PAS w/ diastase ctrl

Stains
Substance Stained
(+) Color/Result
CHO, Glycogen, Mucins,
PAS (+):Magenta red
Bacteria & Fungi,
basement membrane
Glycogen
Red

Best Carmine

Glycogen

Bright red

Langhans iodine method

Glycogen

Mahogany brown

Alcian blue

Acid mucins

Blue

Alcian Blue-PAS

Any mucins
(acid/neutral)
Acid MPS
Sulfated mucins
Carboxylated mucins
Cryptococcus neoformans
Mucins
Acid mucins

Acid mucin: blue

Neutral mucin: magenta
Sulfated mucins: purple
Carboxylated mucins:
blue
Mucin: red

Acid mucins/MPS
Fungi

Stain
PAS

Gomoris aldehyde fuchsin

stain
Mucicarmine stain
Colloidal (Dialyzed) iron
technique
Acridine orange
Sudan black
Sudan IV (Scharlach R)

Lipids
Lipids (TAG)

Oil red O
Osmium tetroxide
Nile blue sulfate method

Lipids
Lipids
Neutral fat
Cholesterin esters
Cholesterin fatty acids
Fatty acids & soap
Cerebrosides

Brilliant red
Black
= Pinkish red
= Light red
= Light red
= Deep blue to violet
= Light blue

Toluidine blue-acetone mtd

Sulfatide

Borohydride-PeriodicSchiff method
Alkaline fast-green method

Gangliosides

Metachromatic redbrown or yellow

Red

Peracetic acid-Alcian blue

Sakaguchis test
Gomori calcium method
Gomori lead method
Lead method
Metal precipitation
Calcium cobalt method
-naphthyl acetate method

Avoid Ehrlichs
hematoxylin

Dark blue
Acid MPS: black
Fungi: greenish red
fluorescence
Blue black
red

Histones
Protamines
Cystine
Cysteine
Arginine
Alkaline phosphatase
Acid phosphatase
5-nucleotidase
ATPase
ATPase
Nonspecific esterase

Comments
Basic fuchsin: essential
component of Schiff
reagent
Method of choice for
glycogen staining
Selective & highly
specific for glycogen
Obsolete
Not specific for glycogen
Avoid celloidinization of
slides
Avoid Ehrlichs
hematoxylin

Green

Lasts for only 2 hrs

Most commonly used

stain
Nile blue: preliminary
indicator of the type of
lipid present
-Red oxazone (dissol.
neutral lipids)
-Blue oxazone (reacts w/
PL and FFA)

Fast green stains basic

groups in tissues

Blue-green
Orange-red
Brownish-black
Black
Blackish brown deposits
Dark brownish-black ppt
Cobalt phosphate ppt
Reddish brown

Uses Milton reagent

Substrate: -glyceroPO4
For skel. muscle biopsies
For skel. muscle biopsies
lec.mt 04 |Page | 282

Stain
Indoxyl acetate method
Tetrazolium method
Feulgen technique

Substance Stained
Nonspecific esterase
Monoamine oxidase
DNA

(+) Color/Result
Blue
Bluish black
Red-purple

Methyl green-pyronin

RNA
DNA
Reticulin fibers

RNA (nucleoli): red

DNA (chromatin): green
black

Collagen
Muscle, cytoplasm, RBC,
fibrin
Collagen & mucus
Muscle, RBC & keratin
Collagen fibers,
cytoplasm, fibroglia
fibrils, axon cylinders,
neuroglia
Elastic fibers
RBCs, myelin sheets
CT
Glomerular basement
membrane
Amyloid & mucous
colloid
Elastic fibers
Elastic fibers
Elastic fibers
Elastic fibers
Fibrin & CT
RBC
RBCs
Muscle
Collagen
Fibrin
Fibrin, muscle striations,
neuroglia, amoeba
RBCs
Myelin
Collagen, osteoid,
cartilage, elastic fibers
Amyloid
Amyloid

= Pink/deep red
= Yellow

Amyloid

Yellow fluorescence

Muscle fibers
Collagen
Muscles, RBC
Collagen

= Red
= Green
= Red
= Yellow

Gomoris silver
impregnation stain
Van Giesons stain
Massons trichrome stain
Mallorys aniline blue

Azocarmine

Weigerts
Verhoeffs
Taenzer-Unna-Orcein mtd
Krajians technique
Martius-Scarlet-Blue

Mallorys PTAH

Congo red
Methyl violet-crystal violet
method
Thioflavin-T fluorescent
staining
Modified Gomoris
Trichrome stain
Lissamine fast red

= Blue
= Red
= Red

Comments
Most reliable & specific
histochemical staining
technique for DNA
Contains Schiffs reagent
Reticulin = Argyrophilic
(silver stain)
Contains acid fuchsin &
picric acid

(-) Fuchsin: Excellent &

colorful method of
demonstrating CT fibers

= Pale pink/yellow
= Yellow

= Deep blue
Dark-blue/blue-black
Black
Dark-brown
= Bright red
= Dark blue
= Orange-yellow
= Yellow
= Red
= Blue
= Red
= Dark blue

Heidenhains
modification of Mallorys
aniline blue stain

Rapid method
Early fibrin = yellow
Old fibrin = blue

= Blue
= Lighter blue
= Deep brownish-red
Red
Purplish red

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Stain
Schmorls Picro-Thionin
method

Substance Stained
Lacunae & canaliculi
Bone matrix

Bielschowskys technique

Neurofibril, axons &

dendrites
Neuroglia & collagen
Bodians stain
Nerve fibers & nerve
endings
Sevier-Munger technique
Peripheral neuritis
Axons
Myelin sheath
Neuritic plaques &
tangles
Argentaffin granules
Toluidine blue
Nissl granules & nucleoli
Polychrome methylene blue Nissl granules & nucleoli
Thionine
Nissl granuls & nucleoli
Cresyl fast violet
Nissl substance
Neurons
Weigert-Pal technique
Myelin sheath
Luxol fast blue
Myelin
Weils method
Myelin
Cajals gold sublimate
Astrocytes
method
Perls Prussian blue
Hemosiderin
Gomoris Prussian blue
Iron pigments
Turnbulls blue
Ferrous iron
(Hemosiderin)
Benzidine-nitroprusside
Hemoglobin & oxidase
stain
granules
Mod. Fouchets technique
Bile pigments
Gmelin technique
Bile & hematoidin
Steins iodine test
Bile pigments
Schmorls ferric
ferricyanide method
Gomoris aldehyde fuchsin
Mallorys fuchsin stain
Masson Fontana technique

Von Kossas silver nitrate

method
Lindquist modified
rhodanine technique

(+) Color/Result
= Dark brown-black
= Yellow/brownishyellow
= Black on a grayish BG

Comments

= Lightly stained
Diagnosis of Alzheimers
disease
= Black
= Black
= Light brown
= Black
= Black
Deep blue
Deep blue
Purple
= Purple-dark blue
= Pale purple blue
Blue black
Blue-green
Black
Black on a light
brownish BG
Deep blue
Bright blue
Blue

Nissl granules: a.k.a.

Tigroid substances

Dark blue
Emerald to blue green
Blue-purple then green
Depend on the oxidation
of the pigment to green
biliverdin by iodine

Bile, lipofuscins,
melanin, argentaffin
cells, chromaffin, thyroid
colloid
Lipofuscin
Hemofuscin
Melanin
Argentaffin cell granules

Dark blue

Calcium

Black

Copper

Red to orange-red

Purple
Red
= Black
= Black

Argentaffin reaction:
melanin reduces
ammoniacal silver
solutions w/o use of a
reducer

lec.mt 04 |Page | 284

Stain
Gram-Twort stain

Brown & Brenn method

Wade-Fite technique
Toluidine blue

Substance Stained
Gram (+) organisms
Gram (-) organisms
RBCs
Elastic fibers
Gram (+) bacteria
Gram (-) bacteria
M. leprae
H. pylori

Cresyl violet acetate mtd

Dieterle method

H. pylori
L. pneumophila &
spirochetes
Levaditis method
Spirochetes
Modified Steiner & Steiner
Spirochetes, Donovan
technique
bodies, fungi, bacteria
Warthin-Starry method
Spirochetes
Grocott Methenamine Silver Fungi

Lendrums phloxinetartrazine method

Orcein method
Giemsa stain

In situ hybridization
PCR
Chondrocalcinosis
Kardasewitsch method
0.1% urea + 5% NaSO4
Metastasis
Degree of localization
Dunn-Thompson
K2MnO4
H2 O 2
Hellys
Formalin ammonium
bromide
Alcohol as 1 fixative
Glutaraldehyde
Carnoys
Orths
Zenkers
Formaldehyde
Ethanol

Mucin & glycogen

Mycelia & hyphae
RBCs
Viral inclusions

(+) Color/Result
= Blue-black
= Pink-red
= Green
= Black
= Blue
= Red
Golden yellow
Dark blue against blue
BG
Blue-violet
Dark brown to black

Comments

Black on a yellowish BG
Black
Black
= Sharply outlined I
black
= Gray-black
= Old rose
= Yellow
Bright red

HBsAg
Brown-black
Bacteria
= Blue
Recommended for blood
Mast cell granules
= Deep blue
and BM parasites,
Eosinophilic granules
= Red
inclusion conjunctivitis,
Nuclei
= Blue
Toxoplasma, spirochetes
Cytoplasm
= Pink
& other bacteria
Most sensitive technique for identifying DNA
DNA amplification
Pseudogout
Pigment removal
70% ETOH + 28% NH3 water
Remove yellow color of HNO3
Most definitive of malignancy
Most reliable indicator of prognosis of malignant tumors
Hgb = emerald green
Removes excess melanin
Contains formalin, K2CrO4 and HAc
Fixative for CNS (gold/silver stain)
Increased tissue shrinkage
Not satisfactory for PAS
Nonaqueous fixative
Pheochromocytoma
PTAH for cross-striations
Wash tissue in water after fixation in Zenkers
Combines w/ amino group
Nonadditive fixative
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pH >6.0 (formalin)
Universal solvents
Soft paraffin
Weigerts hematoxylin
Natural resins (mounting)
Formalin-alcohol
Churukian-Schenk
technique
Masson-Fontana
Muscle biopsies
Paraffin sections
Zambonis PAF
Glutaraldehyde
10% NBF
Paraformaldehyde
Warthin-Starry stain
Iris diaphragm
Substage condenser
Pathology
Cornelius Celsus
Littoral cells
Hoffbauer cells
Cancer
Biohazards
Mercuric chloride
According to the presence
of a mordant
According to the presence
of differentiator
According to the resultant
color
Vital staining

Neurons

Prevent brown microcrystalline deposits in H & E stain

Both dehydrates & clears
For thick sections
Not easily decolorized w/ acidic solutions
Inherently acidic
Microincineration
Substance that can bind silver but need a chemical reducer
Substance that can both bind & reduce silver
Isopentane at -150C
If isopentane is low, dust muscle w/ talc then freeze in liquid nitrogen
Naphthol AS-D chloroacetate esterase
Specimens may remain indefinitely
Specimens may be removed after 2-4 hrs
pH 7.2-7.4
Pure polymer of formaldehyde
Calibrate pH meter to 7.0
Increase amount of light
Adjust to focus the image of the substage diaphragm
Greek: Pathos = suffering
1st to describe the 4 signs of inflammation
Splenic macrophages
Placental macrophages
Latin: Cancrum = crab
Infectious agents
Contaminated solutions
Contaminated specimens
Corrode all metals except for the nickel alloy Monel
Methods of Staining
a. Direct staining = w/o mordant
b. Indirect staining = w/ mordant: serves as a link/bridge between the tissue &
the dye
a. Progressive = w/o differentiator/decolorizer
b. Regressive = w/ differentiator/decolorizer
*1 stain = acidic (decolorizer: basic)
*2 stain = basic (decolorizer: acidic)
a. Orthochromatic = ortho: correct/same | same color = dye & tissue
b. Metachromatic = meta: after/change | different color = dye & tissue
Selective staining of living cells
a. Intravital stain = injection of dye animal body
- Ex. Lithium, Carmine, India ink
b. Supravital stain = staining of cells immediately after removal from the animal
body. Examples are:
- Neutral red = Best vital dye
- Janus green = mitochondria
- Trypan blue
- Nile blue
- Thionine
- Toluidine blue
Functional cells of the CNS
Nerve fibers:
a. Dendrites (Greek: Tree) = conduct impulses to the cell body
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b. Axons = conduct impulses away from the cell body

Criteria for the diagnosis of Marked progesterone effect
normal pregnancy
At least 50% of intermediate cells in clusters
At least some typical pregnancy cells present
<30% of matured superficial cells
Doderlein-filled dirty BG
Staining solutions used in
a. Hematoxylin = dark purple to black
Paps staining method
b. OG-6 = orange w/ a hint of green
(Macroscopic)
c. EA 36/50 = olive green w/ a hint of brown & red
EA 36/50
Components:
a. Eosin Y
b. Bismarck brown
c. Light green SF
# in EA designates the proportion of SF
Affinity:
OG-6
Matured superficial cells
Keratinizing malignant cells
Cytoplasmic:
Bright orange to yellow orange
Affinity:
EA 36/50
Immature vaginal cells (parabasal, intermediate)
Cytoplasm color:
Transparent blue to gray to brown hue
Presently, the Bethesda system divides squamous cell abnormalities into 4 categories:
ASCUS
Atypical cells of undetermined significance
L-SIL
Low-grade squamous intraepithelial lesion
H-SIL
High-grade squamous intraepithelial lesion
SCC
Squamous cell carcinoma
Description
Bethesda 2001
Papanicolau
Normal
(-) for intraepithelial lesion/malignancy
Class I
Atypical
ASCUS
Class II
HPV
L-SIL
Class II
Mild dysplasia
L-SIL
Class II
Moderate dysplasia
H-SIL
Class III
Severe dysplasia
H-SIL
Class III
Carcinoma in situ
H-SIL
Class IV
Invasive carcinoma
Invasive carcinoma
Class V
Microtome
Usual
Description
Tissues embedding in
Microtome
Knives
Length
Plane concave
25 mm One side flat, other Less concave: celloidin-impregnated tissues Sliding
is concave
More concave: paraffin embedded tissues

Biconcave

120 mm

Plane wedge

100 mm

Carmine
Best Carmine
Mucicarmine

Both sides are

Paraffin
concave
Both sides are
Frozen sections
straight
Hard, tough tissue specimen (paraffin)
Chromatin stain
Carmine + Aluminum chloride = For glycogen
Carmine + Aluminum hydroxide = For C. neoformans and mucin

Base-sledge
Rotary
Rocking
Rotary
Sliding
Base-sledge

lec.mt 04 |Page | 287

Picrocarmine
Carmine + Picric acid = for neuropathological studies
Dukes staging for neoplasia One of the most frequently applied for staging individual tumors
of the rectum
Biopsy
Biopsy
Excision and exam (living subject)
Preferred: perform the biopsy at the periphery of the tumor (advancing tumor
margin)
Types of Biopsy
Exfoliative cytology
Desquamated cells
Sex hormonal status in females
Sex chromatin phenotype
Excisional biopsy
Complete removal of a lesion
Most reliable
Incisional biopsy
Removal of part of a lesion/small piece of tumor directly incising the tumor
capsule
Preferred for large tumors that cant be excised completely
Needle biopsy
Aspiration of fluid
Bite biopsy
Small pcs of tumor are removed w/ special forceps
Cutaneous biopsy
Skin fragments
Punch biopsy
For specimens >2mm embed in a single paraffin block
Shave biopsy
Curettage specimens
Wedge biopsy
Specimen is subdivided w/ a razor blade
Marginal excision
Shell-out end

lec.mt 04 |Page | 288